Integration of bioinformatics analysis, molecular docking and animal experiments to study the therapeutic mechanisms of berberine against allergic rhinitis

Allergic rhinitis is a prevalent inflammatory condition that impacts individuals of all age groups. Despite reports indicating the potential of berberine in alleviating allergic rhinitis symptoms, the specific molecular mechanisms and therapeutic targets of berberine remain unclear. This research aims to explore the pharmacological mechanism of berberine in the treatment of allergic rhinitis through bioinformatic analyses and experimental validation. The research utilized public databases to identify potential targets of berberine. Furthermore, differentially expressed genes (DEGs) related to allergic rhinitis were pinpointed from the GSE52804 dataset. Through bioinformatics techniques, the primary targets were discovered and key KEGG and GO-BP pathways were established. To confirm the therapeutic mechanisms of berberine on allergic rhinitis, an OVA-induced allergic rhinitis model was developed using guinea pigs. We identified 32 key genes responsible for the effectiveness of berberine in treating allergic rhinitis. In addition, five central genes (Alb, Il6, Tlr4, Ptas2, and Il1b) were pinpointed. Further examination using KEGG and GO-BP pathways revealed that the main targets were primarily involved in pathways such as NF-kappa B, IL-17, TNF, and inflammatory response. Molecular docking analysis demonstrated that berberine exhibited strong affinity towards these five key targets. Furthermore, the expression levels of IL-6, TLR4, PTGS2, and IL-1β were significantly upregulated in the model group but downregulated following berberine treatment. This research has revealed the mechanism through which berberine combats allergic rhinitis and has identified its potential to regulate pathways linked to inflammation. These discoveries provide valuable insights for the development of novel medications for the treatment of allergic rhinitis.

www.nature.com/scientificreports/Berberine, derived from Rhizoma coptidis (Huanglian), is a key bioactive compound 9 .Recent research has shown that berberine possesses pharmacological properties that target a range of illnesses, such as neurological, cardiovascular, metabolic, gastrointestinal, and cancer-related disorders 10 .Berberine has been shown to inhibit the activation of the NLRP3 inflammasome through mitophagy 11 .Additionally, berberine has garnered attention for its potential in treating respiratory conditions.For instance, berberine demonstrates a beneficial impact on ovalbumin-induced asthma by suppressing the NF-кB signaling pathway 12 .Research has indicated that berberine can alleviate chronic obstructive pulmonary disease induced by cigarette smoke extract by modulating the TGF-β1/Smads signaling pathways 13 .Moreover, berberine mitigates bleomycin-induced pulmonary fibrosis by blocking NF-кB-mediated TGF-β activation 14 .A prior study indicated that berberine has the potential to reduce allergic inflammation in a mouse model of allergic rhinitis 15 .Nevertheless, the specific mechanism by which berberine acts against allergic rhinitis remains unclear.
Network pharmacology represents an innovative strategy for identifying key drug targets and elucidating the underlying mechanisms associated with human diseases 16 .In the present study, network pharmacology was utilized to investigate the molecular mechanisms responsible for the therapeutic effects of berberine in the treatment of allergic rhinitis.The research methodology is visually presented in Fig. 1.

Collecting potential targets of berberine and allergic rhinitis
SwissTargetPrediction is an online tool designed to forecast the primary protein targets of small compounds 17 .The Comparative Toxicogenomics Database (CTD) contains vast collections of selected gene-chemical, genedisease, and chemical-disease associations 18 .Target collection of berberine was performed using the SwissTar-getPrediction (http:// www.swiss targe tpred iction.ch/, assessed on 16 May 2023), and CTD (https:// ctdba se.org/, assessed on 16 May 2023).The Gene Expression Omnibus (GEO) serves as a global public repository where vast amounts of next-generation sequence functional genomic data and high-throughput microarray are deposited by researchers worldwide 19 .The GSE52804 dataset was downloaded from the GEO database (https:// www.ncbi.nlm.nih.gov/ geo/, assessed on 17 May 2023) and used to collect pathological genes of allergic rhinitis.The GSE52804 dataset comprised 3 control group samples (mice challenged with saline) and 3 ovalbumin group samples (mice challenged with ovalbumin).Differential gene expression analysis between control and allergic rhinitis was conducted using the "limma" package (v 3.22.7),with a significance threshold of p < 0.05 and |log fold change| ≥ 0.5.Intersection genes targeted by berberine in allergic rhinitis were identified using the Venn tool.The results were visualized using volcano plots and heat maps generated by the "ggplot2" R package (v 3.3.6).

Molecular docking analysis
The current study utilized molecular docking to investigate the potential binding mode of active compounds with a large molecular receptor.Hub genes were chosen for the docking analysis, and the 3D structures of target proteins (Alb, Il6, Il1b, Tlr4, and Ptgs2) were sourced from the PDB database (https:// www.rcsb.org, assessed on 20 May 2023) with a crystal resolution < 3 Å.Initially, PyMOL software was employed to eliminate solvents and organics from the protein structures.Subsequently, AutoDock software (v 1.5.6) was utilized for the molecular docking analysis, with the results visualized using PyMOL software (v 1.0.0).

Establishment of ovalbumin (OVA)-induced allergic rhinitis model
40 guinea pigs (210-270 g and 6-7 weeks old) were acquired from the Guangdong Institute for Drug Control.The animals were provided with unlimited access to tap water and food, and were housed in a climate-controlled environment (with a relative humidity of 45-65% and a temperature range of 21-25 °C) under a 12-h light/dark cycle.The experimental procedures involving the animals were approved by the Ethics Committee of Fujian Provincial Government Hospital and were carried out in accordance with the ARRIVE guidelines.
Following a one-week acclimation period, the animals were divided into five groups, each consisting of 8 members: the normal control group (NC), the group with OVA-induced allergic rhinitis (OVA), the OVA group of guinea pigs treated with berberine at a dosage of 25 mg/kg (OVA + LBer), the OVA group of guinea pigs treated with berberine at a dosage of 50 mg/kg (OVA + MBer), and the OVA group of guinea pigs treated with berberine at a dosage of 100 mg/kg (OVA + HBer).The allergic rhinitis model was established by sensitizing and stimulating the guinea pigs with OVA, following a method described in a previous study 22 .In short, the guinea pigs in the OVA group were sensitized by receiving OVA (25 μg/guinea pig) injections into their peritoneal cavity three times per week for a period of two weeks.Following this initial sensitization phase, a solution containing OVA (100 μg/guinea pig) was dropped daily into the nasal passages of the guinea pigs for a week using micropipettes.The NC group received repeated sensitization with normal saline.Berberine (25, 50, or 100 mg/kg body weight) was orally given to guinea pigs with OVA-induced allergic rhinitis daily from day 21 to day 28.The dosage selection was based on a previous study 23 .The NC and OVA groups received only saline during the same procedure.

Evaluation of rhinitis symptoms
Following the final treatment, the animals were placed in individual cages for a 30-min observation period, during which the number of times the guinea pigs rubbed and sneezed was documented.

Quantification of levels of inflammatory cytokines and OVA-specific IgE in the serum
The guinea pigs were anesthetized with pentobarbital sodium (40 mg/kg) intraperitoneally 24 h after symptom evaluation.Blood was collected via intraorbital venipuncture, followed by serum separation through centrifugation at 1000g for 10 min.The levels of IL-6, IL-1β, IL-17, TNF-α, and OVA-specific IgE in the serum were quantified using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (ThermoFisher, MA, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)
Nasal mucosa tissues were processed to extract total RNA using TRIzol Reagent (Invitrogen, CA, USA) following the manufacturer's instructions.The purified RNA (2 µg) was converted into cDNA with cDNA synthesis kits (Invitrogen, CA, USA), followed by qRT-PCR analysis on a StepOne Real-time PCR system (Applied Biosystems, CA, USA).mRNA expression levels were quantified using the 2 -ΔΔCt method with the primers listed in Table S1.www.nature.com/scientificreports/

Statistical analysis
R software (v 4.2.1) was utilized for the analysis and visualization of bioinformatics data.The limma package (v 3.52.2) was employed to identify DEGs, while enrichment analysis was conducted with the clusterProfiler package (v 4.4.4).The data from the experiment was analyzed using GraphPad Prism software (v 5.0.1).Results were presented as mean ± standard deviation (SD).Data between the two groups were compared using a t-test.
A statistically significant difference was indicated by p < 0.05.

Ethics approval and consent to participate
The animal experimental procedure received ethical approval from the Ethics Committee of Fujian Provincial Government Hospital and was conducted in adherence to the ARRIVE guidelines.All methods were performed in accordance with the relevant guidelines and regulations.

Screening potential targets of berberine in the treatment of allergic rhinitis
Figure 2A illustrates the utilization of the Swiss Target Prediction and Comparative Toxicogenomics Database for the collection of 523 target genes associated with berberine.Additionally, 837 DEGs were extracted from the GSE52804 dataset.Subsequently, 32 genes were identified as potential targets of berberine in the treatment of allergic rhinitis through the intersection analysis (Fig. 2A and Table 1).The gene volcano plot and heat map depicting these 32 intersection genes are depicted in Fig. 2B,C.

Analysis of the PPI network and identification of primary clusters
Figure 3A illustrates the utilization of Cytoscape software in constructing a PPI network to visualize the interactions among the 32 overlapped genes.Furthermore, the MCODE plugin was employed to pinpoint 14 genes (Cyba, Nfkbia, Fos, Mpo, Il6, Il1b, Sele, Vcam1, F2, Tlr4, Alb, Egr1, Smad3, and Ptgs2) forming a primary cluster (Fig. 3B), potentially representing a crucial regulatory network for berberine in treating allergic rhinitis.

KEGG and GO-BP enrichment analyses
Enrichment analyses were conducted to further explore the biological functions of the potential genes of berberine in relation to allergic rhinitis.As shown in Fig. 4 and Table 2, KEGG enrichment indicated that the 14 core genes were mainly enriched in the TNF signaling pathway (Fig. S2), Leishmaniasis, AGE-RAGE signaling pathway in diabetic complications, Chagas disease, Malaria, IL-17 signaling pathway, NF-kappa B signaling pathway (Fig. S1), Toll-like receptor signaling pathway, Th17 cell differentiation, and African trypanosomiasis, etc.As presented in Fig. 5 and Table 3, GO-BP enrichment analysis revealed that the 14 core genes were significantly involved in positive regulation of nitric oxide biosynthetic process, positive regulation of nitric oxide metabolic process, inflammatory response, regulation of nitric oxide biosynthetic process, regulation of nitric oxide metabolic process, nitric oxide biosynthetic process, nitric oxide metabolic process, reactive nitrogen species metabolic process, interleukin-1 beta production, and positive regulation of response to external stimulus, etc.   www.nature.com/scientificreports/GSEA Enrichment analysis using GSEA was conducted to further assess the associated pathways involved in the development of allergic rhinitis.As shown in Fig. 6, the findings revealed that the DEGs were significantly enriched in inflammation-related and immune-related pathways, including KEGG toll-like receptor signaling pathway, WP photodynamic therapy induced NFKB survival signaling, WP TNF-α signaling pathway, KEGG cytokinecytokine receptor interaction, REACTOME adaptive immune system, and REACTOME cytokine signaling in immune system.

Hub genes identification
The results from Table 4 show that nine topological analysis algorithms in cytoHubba selected the top 10 genes.Subsequently, the R package "UpSet" was utilized to pinpoint five hub genes-Alb, Il6, Il1b, Tlr4, and Ptgs2 (Fig. 7A).Additionally, a heatmap illustrating the mRNA expression levels of these five hub genes in the GSE52804 dataset was created for visualization purposes (Fig. 7B).www.nature.com/scientificreports/

Berberine inhibited hub genes expression in OVA-induced allergic rhinitis
The validation of molecular docking results was conducted through qRT-PCR analysis.Figure 11 illustrates that treatment with berberine (100 mg/kg) led to a significant decrease in the expressions of IL-6, IL-1β, TLR4, and PTGS2 in the OVA group, while the expression of ALB showed a notable increase in the OVA group (p < 0.01).

Discussion
Allergic rhinitis is a chronic respiratory tract disease mediated by immunoglobulin E, characterized by symptoms such as nasal congestion and frequent sneezing 24 .This condition not only significantly impacts the patient's daily life and productivity but also worsens other symptoms like asthma 25 .Despite extensive research on treatments for rhinitis, there remains a significant lack of consistently effective treatments for a large number of patients 26,27 .
Chinese traditional medicine has a long history of effectively treating allergic rhinitis symptoms 28,29 .Berberine, an  www.nature.com/scientificreports/alkaloid derived from Rhizoma coptidis, is known for its diverse pharmacological benefits, including anti-allergic, anti-inflammatory, and anti-tumor properties 30 .Berberine has also been shown to inhibit allergic symptoms in the ovalbumin-induced allergic rhinitis model 31 .Nevertheless, the exact mechanism through which berberine acts against allergic rhinitis remains unclear.Utilizing network pharmacological analysis is an innovative method for systematically exploring the therapeutic mechanisms of active components in diseases.Hence, this approach was employed to investigate potential interactions between target genes and active ingredients, thereby aiding in drug development 16 .The current study utilized a network pharmacology approach to uncover the potential mechanism responsible for the therapeutic benefits of berberine in treating allergic rhinitis.Initially, 14 hub potential therapeutic targets were identified through bioinformatics analysis of the GSE52804 dataset and berberine targets.Subsequent enrichment analyses were carried out utilizing these therapeutic targets.GO-BP, KEGG, and GSEA results indicated that these targets were mainly enriched in inflammation-and immune-related pathways, the important etiological pathways of allergic rhinitis, such as inflammatory response, NF-kappa B signaling pathway, TNF signaling pathway, adaptive immune system, and cytokine signaling in immune system, etc. NF-kappa B signaling pathway is implicated in the pathologic processes of rhinitis 32 .The development of chronic inflammation in rhinitis is triggered by the intricate interplay of various cytokines, with the activation of the NF-kappa B signaling pathway driving this pathological process 33 .Activation of the NF-kappa B signaling pathway could promote IL-6 and IL-8 expression in chronic nasal sinusitis 34 .Moreover, overactive NF-kappa B signaling pathway was found to contribute to the upregulation of ICAM-1 expression, which is closely linked to the advancement of allergic rhinitis 35 .The levels of NF-kappa B expression from chronic rhinosinusitis patients were higher than that of healthy people 36 .TNF plays a crucial role as a cytokine involved in a variety of biological functions, including regulating cell death, proliferation, survival, immune responses, and inflammation 37 .TNF has the ability to induce the expression of NF-kappa B and modulate immune responses, which are crucial in the development of airway inflammation 38 .Abnormal TNF signaling is involved in the pathogenesis of various diseases, including allergic rhinitis 39,40 .Additionally, the TNF signaling pathway and the cytokine-cytokine receptor interaction were identified as key KEGG pathways in a mouse model of allergic rhinitis 41 .Berberine has been shown to deactivate the NF-kappa B signaling pathway and alleviate airway inflammation in a rat model of asthma 12 .Our analysis suggests that the TNF signaling pathway and NF-kappa B signaling pathway may be the underlying therapeutic mechanisms of berberine in treating allergic rhinitis.
Ultimately, 5 key genes (Alb, Il6, Il1b, Tlr4, and Ptgs2) were pinpointed using the R package "UpSet" and validated through molecular docking to further elucidate the intricate molecular interactions related to therapeutic targets.Among them, Il6 and Il1b are known as proinflammatory cytokines.Previous studies have indicated the involvement of cytokines in the pathogenesis of allergic rhinitis 42,43 .Il6 plays a crucial role in the development of allergic rhinitis, and its levels escalate as the disease advances 44 .Serum Il1b was identified as a potential biomarker for severe persistent allergic rhinitis 45 .Overexpression of Tlr4 is observed in individuals suffering from persistent allergic rhinitis, contributing to the advancement of allergic inflammation in this condition 46 .www.nature.com/scientificreports/Additionally, in a mouse model of allergic rhinitis, the Tlr4 antagonist was found to reduce lung injury by suppressing inflammatory monocytes 47 .Ptgs2 (alias is Cox2) has been reported to play an important role in the pathophysiology of airway inflammatory response 48 .The enrichment analysis findings in this study revealed the involvement of five hub genes in the TNF and NF-kappa B signaling pathways.Moreover, molecular analysis and animal experiment were conducted to validate the network pharmacology results, demonstrating that berberine displayed strong binding affinity to Alb, Il6, Il1b, Tlr4, and Ptgs2.While this study employs molecular docking to predict the interactions between berberine and the protein of hub genes identified in allergic rhinitis, it is imperative to acknowledge the inherent limitations and challenges of this approach: 1.Molecular docking analysis relies on computational models to predict molecular interactions, which may introduce errors and uncertainties due to the accuracy of the model and parameter settings.2. Molecular docking analysis is typically based on static structures, while molecules in biological systems are dynamic and constantly changing, potentially leading to discrepancies between the predicted interactions and the actual biological interactions.3. Molecular docking analysis often requires a large amount of computational resources and time, which may limit the scope and scale of the analysis.

Conclusion
In general, the current research effectively emphasized potential pharmacological mechanisms and therapeutic targets of berberine in treating allergic rhinitis through network pharmacology and molecular docking analyses.Additionally, the therapeutic impact of berberine on allergic rhinitis may occur by influencing inflammationrelated pathways such as TNF and NF-kappa B signaling pathways.This study also suggested that the integration

Figure 1 .
Figure 1.Workflow of the present study.

Figure 2 .
Figure 2. Collecting potential targets of berberine in the treatment of allergic rhinitis.(A) Venn diagram of DEGs and berberine-related genes.(B) The gene volcano map of DEGs.Green dots represented the down-regulated genes, red dots represented the up-regulated genes, and gray dots represented genes with no significant differences.(C) Clustered heat map of the 32 intersection genes.

Figure 3 .
Figure 3. Analysis of PPI network and clusters.(A) PPI network of 32 intersection genes visualized by Cytoscape software.(B) Primary clusters are constructed based on the MCODE plugin.

Figure 4 .
Figure 4. KEGG enrichment analysis.The Sankey map showed the relationship between targets and the top 10 KEGG pathways (left).The bubble diagram depicted the top 10 KEGG pathways (right).

Figure 5 .
Figure 5. GO-BP enrichment analysis.The Sankey map showed the relationship between targets and the top 10 GO-BP terms (left).The bubble diagram depicted the top 10 GO-BP terms (right).

Figure 9 .
Figure 9. Berberine improved rhinitis symptoms in OVA-induced allergic rhinitis.The sneezing (A) and rubbing (B) times of animals were recorded.## p < 0.01, the OVA group compared to the NC group; **p < 0.01, the therapeutic group compared to the OVA group.Allergic rhinitis was induced through an intraperitoneal injection of OVA (25 µg/guinea pig) three times a week for two weeks.The guinea pigs from the OVA group were treated with different doses of berberine (LBer: 25 mg/kg body weight; MBer: 50 mg/kg body weight; HBer: 100 mg/kg body weight).

Figure 10 .
Figure 10.Berberine alleviates inflammatory response in OVA-induced allergic rhinitis.The TNF-α (A), IL-6 (B), IL-1β (C), IL-17 (D), and IgE (E) levels in serum.## p < 0.01, the OVA group compared to the NC group; **p < 0.01, the therapeutic group compared to the OVA group.Allergic rhinitis was induced through an intraperitoneal injection of OVA (25 µg/guinea pig) three times a week for two weeks.The guinea pigs from the OVA group were treated with different doses of berberine (LBer: 25 mg/kg body weight; MBer: 50 mg/kg body weight; HBer: 100 mg/kg body weight).

Figure 11 .
Figure 11.Verification of IL-6 (A), IL-1β (B), TLR4 (C), PTGS2 (D), and ALB (E) expression in OVA-induced allergic rhinitis.## p < 0.01, the OVA group compared to the NC group; **p < 0.01, the therapeutic group compared to the OVA group.Allergic rhinitis was induced through an intraperitoneal injection of OVA (25 µg/ guinea pig) three times a week for two weeks.The guinea pigs from the OVA group were treated with different doses of berberine (HBer: 100 mg/kg body weight).

Table 1 .
32 intersection genes of berberine and DEGs in GSE52804.

Table 2 .
Top 10 KEGG pathways of berberine against allergic rhinitis.

Table 3 .
Top 10 GO biological processes of berberine against allergic rhinitis.

Table 4 .
Top 10 genes by 9 ranked methods in cytoHubba.

Table 5 .
Binding energies of berberine to the hub target proteins.